Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
Perlmutter Cancer Center, NYU Langone, New York, USA.
BMC Res Notes. 2024 Sep 16;17(1):267. doi: 10.1186/s13104-024-06927-0.
Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps-(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.
到目前为止，已经开发出了多种单细胞分析技术，但我们仍然缺乏一种相对简单的工具，可以从同一细胞中以全转录组和全基因组的深度同时评估 DNA 和 RNA。在这里，我们提出了一种从细胞核中分离细胞质 RNA 的更新方法，该方法允许从同一单细胞同时研究 DNA 和 RNA。该方法包括三个步骤-(1)将单个细胞固定在固体基质上，(2)固定化单细胞的低渗裂解，以及(3)分离含有细胞质的水相和固定化核。我们发现，使用我们的方法从单个细胞中提取的 DNA 和 RNA 适用于下游基于测序的应用。我们证明，在 DNA/RNA 分离后获得的转录组和基因组测序数据的覆盖度与未经分离观察到的相似。我们还表明，分离过程不会在观察到的突变负荷或突变谱中产生任何明显的偏差。因此，我们的方法可以作为同时对基因组和转录组进行复杂分析的工具，提供关于体细胞突变与基因表达调控之间关系的必要信息。
